Lu P, Davis B P, Hendrick J, Jeffries T W
Department of Bacteriology, University of Wisconsin-Madison 53706, USA.
Appl Microbiol Biotechnol. 1998 Feb;49(2):141-6. doi: 10.1007/s002530051150.
Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5'-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ "pop-out" cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu- Ura+ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5'-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu- Ura- phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.
为了推进树干毕赤酵母的遗传学研究和木糖代谢的发展，需要对树干毕赤酵母进行转化。为此，我们使用树干毕赤酵母URA3（PsURA3）在树干毕赤酵母ura3突变体中破坏树干毕赤酵母LEU2。选择一株高发酵性的树干毕赤酵母突变体（FPL-DX26）对5'-氟乳清酸具有抗性，从而获得树干毕赤酵母FPL-UC7（ura3-3）。构建了一个URA3:lacZ“弹出”盒，其中PsURA3两侧是来自lacZ阅读框片段的直接重复序列。通过与酿酒酵母LEU2的同源性，从树干毕赤酵母CBS 6054基因组文库中克隆了树干毕赤酵母LEU2基因（PsLEU2），并通过用PsURA3:lacZ盒替换PsLEU2阅读序列构建了一个破坏盒。用该破坏盒转化FPL-UC7（ura3-3），并通过选择亮氨酸缺陷型尿嘧啶原养型表型鉴定出一个位点特异性整合体。通过将细胞接种到5'-氟乳清酸盐上并筛选自发的URA3缺失突变体，从该菌株中回收ura3标记。侧翼PsURA3基因的切除导致亮氨酸缺陷型尿嘧啶缺陷型表型。双营养缺陷型是稳定的，并且可以被基于自主复制序列的质粒携带的PsLEU2或PsURA3高频转化。
